The purpose of this study was to determine whether phosphorylation has an effect on the characteristics of the 60 kD Ro antigen throughout the cell cycle. Cell extracts of synchronized HEp-2 cells were phosphorylated in vitro with exogenous ATP, examined by SDS-PAGE and Western blot, and probed with specific anti-Ro sera. In addition, cellular ATP pools were radiolabelled in vivo with 32P. The presence of the Ro protein was detected with a molecular weight of 60 kD during all phases of the cell cycle, except at the M phase, where it was increased to 65 kD. Phosphorylation of the in vitro and in vivo cell extracts increased the molecular mass to 65 kD. Moreover immunoprecipitation assays demonstrated that Ro is hyperphosphorylated in the M phase. Phosphorylation did not change the recognition pattern of the anti-Ro sera.